Dystrophin-glycoproteins associated in congenital muscular dystrophy: immunohistochemical analysis of 59 Brazilian cases

The congenital muscular dystrophies (CMD) are heterogeneous muscular diseases with early and dystrophic pattern on muscle biopsy. Many different subtypes have been genetically identified and most phenotypes not yet identified belong to the merosin-positive (MP) CMD subgroup. OBJECTIVE: To analyze the immunohistochemical expression of the main proteins of the dystrophin-glycoproteins associated complex in muscle biopsy of patients with different CMD phenotypes, for investigating a possible correlation with clinical and histopathological data. METHOD: Fifty-nine patients with CMD had clinical, histopathological and immunohistochemical data evaluated: 32 had MP-CMD, 23 CMD with merosin deficiency (MD-CMD), one Ullrich phenotype and three Walker-Warburg disease. RESULTS: Dystrophin and dysferlin were normal in all; among the patients with MD-CMD, merosin deficiency was partial in nine who showed the same clinical severity as those with total deficiency; the reduced expression of alpha-sarcoglycan (SG) and alpha-dystroglycan (DG) showed statistically significant correlation with severe MD-CMD phenotype. CONCLUSION: There is a greater relationship between merosin and the former proteins; among MP-CMD patients, no remarkable immunohistochemical/phenotypical correlations were found, although the reduced expression of beta-DG had showed statistically significant correlation with severe phenotype and marked fibrosis on muscular biopsy.

A distrofia muscular congênita (DMC) é doença muscular heterogênea, de início precoce e padrão histopatológico de distrofia. Diversos subtipos foram geneticamente identificados e os fenótipos ainda não identificados pertencem em geral ao subgrupo de DMC merosina-positiva (MP). OBJETIVO: Analisar a expressão imuno-histoquímica das principais proteínas do complexo distrofina-glicoproteínas associadas na biópsia muscular de pacientes com diferentes fenótipos de DMC, a fim de investigar uma eventual correlação com o quadro clínico e histopatológico. MÉTODO: Cinqüenta e nove pacientes com DMC foram avaliados clinicamente e sua biópsia muscular, histopatologica e imuno-histoquimicamente: 32 eram MP, 23 merosina-deficiente (MD), um mostrava fenótipo Ullrich e três síndrome de Walker-Warburg. RESULTADOS: Distrofina e disferlina foram normais em todos; nove pacientes MD apresentavam déficit parcial de merosina, porém com a mesma gravidade clínica daqueles com deficiência total. CONCLUSÃO: A hipoexpressão de a-sarcoglicana (SG) and a-distroglycan (DG) se correlacionou estatisticamente com o grave fenótipo MD, assim indicando maior correlação entre a merosina e as referidas proteínas; entre os pacientes MP, apesar da hipoexpressão de b-DG ter se correlacionado significativamente com fenótipo e histopatologia mais grave, não houve correlação clínica/imuno-histoquímica valorizável.

Ullrich congenital muscular dystrophy and Bethlem myopathy: clinical and genetic heterogeneity

Ullrich congenital muscular dystrophy (UCMD), due to mutations in the collagen VI genes, is an autosomal recessive form of CMD, commonly associated with distal joints hyperlaxity and severe course. A mild or moderate involvement can be occasionally observed. OBJECTIVE: To evaluate the clinical picture of CMD patients with Ullrich phenotype who presented decreased or absent collagen VI immunoreactivity on muscular biopsy. RESULTS: Among 60 patients with CMD, two had no expression of collagen V and their clinical involvement was essentially different: the first (3 years of follow-up) has mild motor difficulty; the second (8 years of follow-up) never acquired walking and depends on ventilatory support. A molecular study, performed by Pan et al. at the Thomas Jefferson University, demonstrated in the first a known mutation of Bethlem myopathy in COL6A1 and in the second the first dominantly acting mutation in UCMD and the first in COL6A1, previously associated only to Bethlem myopathy, with benign course and dominant inheritance. CONCLUSION: Bethlem myopathy should be considered in the differential diagnosis of UCMD, even in patients without fingers contractures; overlap between Ullrich and Bethlem phenotypes can be supposed.

A distrofia muscular congênita (DMC) com hiperextensibilidade articular distal (fenótipo Ullrich) associa-se a mutações nos genes do colágeno VI e corresponde a um grave quadro congênito de herança autossômica recessiva e curso progressivo, ocasionalmente mostrando menor gravidade. OBJETIVO: Avaliar o quadro clínico dos pacientes com DMC tipo Ullrich que apresentam imunoexpressão baixa ou ausente do colágeno VI na biópsia muscular. RESULTADOS: Entre 60 pacientes com DMC, dois mostravam imunomarcação negativa do colágeno VI. Mostravam-se clinicamente essencialmente diferentes: o primeiro, com 8 anos de idade e três de seguimento mostra leve dificuldade motora; o segundo, com 14 anos de idade e 8 de seguimento, não deambula e apresenta insuficiência respiratória. O estudo molecular, realizado na Thomas Jefferson University por Pan et al., revelou no primeiro, no gene COL6A1, mutação típica da miopatia de Bethlem, que tem curso benigno e herança autossômica dominante; e no segundo a primeira mutação de efeito dominante e do gene COL6A1, previamente associado apenas à miopatia de Bethlem. CONCLUSÃO: A miopatia de Bethlem deve constar no diagnóstico diferencial da DMC tipo Ullrich, mesmo na ausência das típicas contraturas dos dedos; pode existir sobreposição dos fenótipos Ullrich e Bethlem.

Biphenylsulfonamide endothelin receptor antagonists. 2. Discovery of 4'-oxazolyl biphenylsulfonamides as a new class of potent, highly selective ET(A) antagonists.

The synthesis and structure-activity relationship (SAR) studies of a series of 4'-oxazolyl-N-(3,4-dimethyl-5-isoxazolyl)[1, 1'-biphenyl]-2-sulfonamide derivatives as endothelin-A (ET(A)) receptor antagonists are described. The data reveal a remarkable improvement in potency and metabolic stability when the 4'-position of the biphenylsulfonamide is substituted with an oxazole ring. Additional 2'-substitution of an acylaminomethyl group further increased the binding activity and provided one of the first subnanomolar ET(A)-selective antagonists in the biphenylsulfonamide series (17, ET(A) K(i) = 0.2 nM). Among the compounds described, 3 (N-(3,4-dimethyl-5-isoxazolyl)-4'-(2-oxazolyl)[1, 1'-biphenyl]-2-sulfonamide; BMS-193884) had the optimum pharmacological profile and was therefore selected as a clinical candidate for studies in congestive heart failure.

Biphenylsulfonamide endothelin antagonists: structure-activity relationships of a series of mono- and disubstituted analogues and pharmacology of the orally active endothelin antagonist 2'-amino-N- (3,4-dimethyl-5-isoxazolyl)-4'-(2-methylpropyl)[1, 1'-biphenyl]-2-sulfonamide (BMS-187308).

Substitution at the ortho position of N-(3,4-dimethyl-5-isoxazolyl) benzenesulfonamide led to the identification of the biphenylsulfonamides as a novel series of endothelin-A (ETA) selective antagonists. Appropriate substitutions on the pendant phenyl ring led to improved binding as well as functional activity. A hydrophobic group such as isobutyl or isopropoxyl was found to be optimal at the 4'-position. Introduction of an amino group at the 2'-position also led to improved analogues. Combination of the optimal 4'-isobutyl substituent with the 2'-amino function afforded an analogue (20, BMS-187308) with improved ETA binding affinity and functional activity. Compound 20 also has good oral activity in inhibiting the pressor effect caused by an ET-1 infusion in rats. Doses of 10 and 30 micromol/kg iv 20 attenuated the pressor responses due to the administration of exogenous ET-1 to conscious monkeys, indicating that the compound inhibits the in vivo activity of endothelin-1 in nonhuman primates.

Exogenous effects and endogenous production of endothelin in cardiac myocytes: potential significance in heart failure.

Increased plasma concentrations of endothelin have been identified in patients and animals with severe congestive heart failure (CHF). However, whether and to what extent increased endothelin (ET) concentrations influence left ventricular (LV) myocyte contractility, ET-receptor subtype density, and endogenous ET production with the development of CHF remains unclear. Accordingly, myocyte contractile function, response to ET, sarcolemmal ET-receptor density, and myocyte ET production were examined in pigs following the development of pacing-induced CHF (240 beats/min, 3 wk, n = 8) and in controls (n = 8). With CHF, plasma ET increased over threefold. In the presence of ET (10-500 pM), myocyte contractility increased in a dose-dependent manner in control myocytes but decreased in CHF myocytes. For example, in the presence of 200 pM ET, velocity of shortening increased by 32.8 +/- 2.3 microns/s in controls but decreased by 8.3 +/- 2.2 microns/s with CHF. LV sarcolemmal ET-receptor density was primarily of the ETA-receptor subtype in controls (96 +/- 1.0%) and was unchanged with CHF. In quiescent myocyte preparations, control myocytes secreted ET (2.29 +/- 0.45 amol.cell-1.h-1), which was similar in CHF myocytes. These findings suggest that the production of ET may have important and potentially differential effects on contractile function with the development of CHF.

Mutational analysis of the endothelin type A receptor (ETA): interactions and model of selective ETA antagonist BMS-182874 with putative ETA receptor binding cavity.

Endothelin (ET) receptor antagonism is a potential therapeutic intervention in the treatment of vascular diseases. To elucidate the mechanism of antagonist-ET receptor complex formation, the interactions of four chemically distinct antagonists were investigated using a combination of genetic and biochemical approaches. By site-specific mutagenesis we previously demonstrated that Tyr129 in the second transmembrane domain was critical for high-affinity, subtype-selective binding to the A subtype of ET (ETA) receptors [Krystek et al. (1994) J. Biol. Chem. 269, 12383-12386]. Affinities of the constrained cyclic pentapeptide BQ-123, the pyrimidinylbenzenesulfonamide bosentan, the indancarboxlic acid SB 209670, and the naphthalenesulfonamide BMS-182874 were decreased 20-1000-fold in Tyr129Ala, Tyr129Ser, and Tyr129His ETA receptor mutants. Substitution of Tyr129 with Phe or Trp did not alter the high-affinity binding of BQ-123, bosentan, or SB 209670. BMS-182874 binding affinity was decreased 10-fold in Tyr129Phe and Tyr129trp ET receptors. These data indicate a role of aromatic interactions in the binding of these antagonists to ETA receptors an, in the case of BMS-182874, also suggested a hydrogen bond with the tyrosine hydroxyl. This hypothesis was supported by structure-activity data with analogs of BMS-182874 that varied the C-5 dimethylamino substituent on the naphthalene ring. Mutation of Asp126 and Asp133 also altered binding of BMS-182874 and C-5 analogs. In all cases, naphthalenesulfonamide binding was more severely affected by mutation of Asp133 than by mutation of Asp126. Phosphoinositide hydrolysis and extracellular acidification rate studies demonstrated the importance of Tyr129 to ETA-mediated signal transduction. On the basis of these data, two plausible models of the docked conformation of BMS-182874 in the ETA receptor are proposed as a starting point for further delineation of interactions that underlie antagonist-ETA receptor complex formation.

Discovery and structure-activity relationships of sulfonamide ETA-selective antagonists.

Random screening of compounds in an ETA receptor binding assay led to the discovery of a class of benzenesulfonamide ligands. Optimization led to the development of 5-amino-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalenesulfonamides which were functional antagonists. Structural features which were important to activity included a 1,5-substitution pattern on the naphthalene ring; a sulfonamide NH with a pK value < 7; an amine, preferably with alkyl substituents, at the 5-position; and methyl groups on both the 3- and 4-positions of the isoxazole.

Cloning and expression of an endothelin receptor subtype B from human prostate that mediates contraction.

Recent evidence suggests a role for endothelin (ET) in contraction of human prostate [J. Urol. 149:495-499 (1993)]. Although both ETA and ETB receptors have been shown to mediate contraction of smooth muscle, the molecular identity of the contractile ETB receptor is controversial. The aim of this study was to examine the receptor subtype that mediates ET-induced contraction in prostate from patients with benign prostatic hyperplasia. Saturation binding with 125I-ET-1 and 125I-ET-3 in prostate stromal cells (PSC) indicated the presence of receptors with subnanomolar affinity for these radioligands, with equivalent receptor densities. Inhibition of specific 125I-ET-1 or 125I-ET-3 binding in PSC revealed a rank order of potency of ET-1 - ET-3 = sarafotoxin S6c >> BQ-123. These data are consistent with a predominance of ETB receptors in PSC. The functional effects of ET stimulation of PSC were examined in a collagen gel contraction assay. ET-1 and ET-3 caused contraction of underlying collagen gel matrices with EC50 values of 0.4 +/- 0.04 and 0.7 +/- 0.2 nM, respectively. To determine the molecular nature of the contractile ETB receptor in PSC, reverse transcription-polymerase chain reactions were conducted with oligonucleotide primers to the 5' and 3' ends of the coding sequence of the full length human ETB receptor. DNA sequence analysis of the 1.3-kilobase DNA product showed 99% homology to other human ETB receptor cDNAs. The encoded protein has a deduced amino acid sequence identical to that of other human ETB receptors, with the exception of two conservative substitutions. Expression of the PSC ETB cDNA in COS-7 cells resulted in a binding profile similar to that observed in parent cells. Polymerase chain reaction analysis revealed the presence of prepro-ET-1 mRNA in PSC. Collectively, these data indicate that PSC from patients with benign prostatic hyperplasia express ETB receptors that mediate ET-induced contraction.

Aspartate mutation distinguishes ETA but not ETB receptor subtype-selective ligand binding while abolishing phospholipase C activation in both receptors.

The endothelin receptors, ETA and ETB, are G protein-coupled receptors (GPCR) that show distinctively different binding profiles for the endothelin peptides and other ligands. We recently reported that Tyr129 in the second transmembrane region (TM2) of the ETA receptor was critical for subtype-specific ligand binding [Krystek, S.R. et al. (1994) J. Biol. Chem. 269, 12383-12386]. Receptor models indicated that aspartic acids located one helical turn above (Asp133) and below (Asp126) Tyr129 in ETA had their side chains directed toward the putative binding cavity. Similarly in ETB, Asp147 and Asp154 are located one turn below and above His150, the residue that corresponds to Tyr129. Asp126 in ETA and Asp147 in ETB correspond to the highly conserved aspartate present in TM2 of many GPCR that has frequently been shown to be crucial for agonist efficacy. Mutagenesis of Asp126 of the human ETA receptor to alanine resulted in an unaltered affinity for ET-1, a 160-fold increase in ET-3 affinity and a decrease in affinity for the ETA selective naphthalenesulfonamide, BMS-182874. ET-1 activation of phospholipase C was abolished. In addition, despite the gain in binding affinity, ET-3 failed to activate phospholipase C, suggesting that Asp126 is required for signal transduction. Mutagenesis of Asp133 to alanine indicated that it was critical only for the binding of BMS-182874. In the ETB receptor, mutation of His150 to alanine or tyrosine indicated that it plays a minor role in ETB subtype-selective ligand binding; mutation of the aspartates in TM2 of ETB did not alter ligand binding. As in the Asp126 Ala ETA variant, ET-1 and ET-3 failed to increase intracellular levels of inositol phosphates in the Asp147Ala ETB mutant. Taken together, these data support the hypothesis that Asp126 and Asp133 flanking Tyr129 in TM2 of the ETA receptor play a role in defining ETA subtype-selective ligand binding but Asp147 and Asp154 that flank the His150 in TM2 of the ETB receptor do not. Furthermore, these data indicate that Asp126 in ETA and Asp147 in ETB are important for transmembrane signaling via phospholipase C.

BMS-182874 is a selective, nonpeptide endothelin ETA receptor antagonist.

BMS-182874 [5-(dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalene sulfonamide] is a recently discovered, low molecular weight, nonpeptide endothelin (ET) receptor antagonist. BMS-182874 competitively inhibited the binding of [125I]ET-1 to ETA receptors in rat vascular smooth muscle A10 (VSM-A10) cell membranes (Ki = 61 nM) and in CHO cells stably expressing the human ETA receptor (Ki = 48 nM), but was a weak inhibitor at ETB receptors (Ki > 50 microM) and non-ET receptors. BMS-182874 inhibited ET-1-stimulated inositol phosphate accumulation (KB = 75 nM) and calcium mobilization (KB = 140 nM) without suppressing the maximal responses in VSM-A10 cells. BMS-182874 was a competitive antagonist of force development elicited by stimulation of ETA, but not other, receptors in isolated blood vessels such as the rabbit carotid artery (KB = 520 nM). The apparent discrepancy between efficacy in cell and tissue models was likely related to the high degree of protein binding exhibited by BMS-182874. When administered either orally (ED50 = 30 mumol/kg) or intravenously (ED50 = 24 mumol/kg) to conscious, normotensive rats, BMS-182874 blunted the pressor response to exogenous ET-1. These data demonstrate that BMS-182874 is a competitive, selective and orally active ETA receptor antagonist that will be useful in understanding the role of ET in normal and disease states.

The receptor binding affinity of monocyclic [Ala3,Xaa11]endothelin-1 analogs correlates with inducible helix length.

Endothelin-1, a bicyclic 21-amino acid peptide with disulfide bridges between cysteines 1 and 15 as well as between cysteines 3 and 11, has been reported to be partially helical based on both CD and NMR data. However, this remains an area of controversy with some claims that CD data indicate no alpha-helical structure (Calas, B.; Harricane, M.-C.; Gulmard, L.; Heitz, F.; Mendre, C.; Chabrier, P.E.; Bennes, R. Peptide Res. 1992, 5, 97) and a recent X-ray crystal structure placing the helix at a different locus (Janes, R.W.; Peapus, D.H.; Wallace, B.A. Structural Biology 1994, 1, 311). The CD studies reported herein indicate that the helical structures reported in NMR studies (e.g. Andersen, N.H.; Chen, C.; Marschner, T.M.; Krystek, Jr. S.R.; Bassolino, D.A. Biochemistry 1992, 31, 1280) apply to pure aqueous media as well. The helix located from Lys9 to the Cys15/His16 juncture is ca 75% populated in pH 4 aqueous buffer. Titration difference CDs reveal that the helix extent increases by one to two residues and that the 'helical conformation' is more completely populated upon addition of TFE to 50+ volume-%. Comparison with a more helical analog suggests that the helix propagates towards (but not to the end of) the C-terminus upon fluoroalcohol addition. A variety of monocyclic derivatives of [Nle7] ET-1 lacking the 3,11-disulfide were evaluated for biological activity and examined by TFE titration difference CD. The series included an Aib11 and a Pro11 analog. The helix promoting Aib analog was the most active while the Pro analog exhibited significantly lower vasoconstrictor activity and binding affinity for the ETA receptor. All of the monocyclic analogs became significantly more helical upon addition of fluoroalcohols. The inclusion of a proline residue at position 11 does not preclude helix formation upon addition of fluoroalcohols. Rather, helix formation is relatively easily induced but limited to a 5 residue span. Apparently this is insufficient to orient required side chains optimally for interaction with the ETA receptor. For the 1,15-monocyclic analogs differing only at position 11, ETA binding affinity and vasoconstrictor potency correlate with the facility which a 7-8 residue long helix can be induced. This presumably includes the segment Glu10-->Cys15 in all cases and may represent the full sequence from Lys9-->His16. CD studies also reveal that the C-terminal fragment of endothelins is not a fully disordered 'random coil' either alone or attached to the endothelin core.

Comparison of a novel ETA receptor antagonist and phosphoramidon in renal ischemia.

Infusion (0.46 mumol/kg/min) of the endothelin (ET)-converting-enzyme inhibitor, phosphoramidon (P), protected function and structure after 30 min renal ischemia in rats more than treatment (5 mumol/kg/min) with the ETA receptor antagonist, BMS-182874 (B). The glomerular filtration rate (GFR; 0.7 +/- 0.12 ml/min) and renal plasma flow (RPF) decreased approximately 40% at 2 h reflow versus controls (C: 1.2 +/- 0.12). B weakly protected the GFR (0.8 +/- 0.07 ml/min); P restored it (1.1 +/- 0.05). Both compounds reduced tubular injury at 2 h reflow; P ameliorated glomerular changes. At 24 h the GFR (0.6 +/- 0.06 ml/min) and RPF decreased 67% versus C (1.8 +/- 0.08). B did not protect the GFR and RPF. P partially protected the GFR (0.9 +/- 0.07 ml/min) but not RPF, and reduced tubular injury. The results suggest that both ETA and non-ETA receptors mediate ET-induced changes in ischemic renal failure.

Mutation of peptide binding site in transmembrane region of a G protein-coupled receptor accounts for endothelin receptor subtype selectivity.

The molecular basis for endothelin (ET) isopeptide selectivity between ETA and ETB receptors was studied by examining ligand binding to several site-specific mutants of the human ETA receptor. Based on a computer-built three-dimensional model of the ETA receptor, five non-conserved amino acids, clustered around the putative ligand binding site, were targeted for mutation to alanine. Expression of the wild-type and mutant ETA receptors in COS-7 cells revealed that the binding profile of one of the ETA mutants, Tyr129-->Ala, was characteristic of the ETB receptor. In the Tyr129-->Ala ETA receptor mutant the affinity of two ETB-selective agonists, endothelin-3 and sarafotoxin S6c, was increased 10-200-fold, whereas that for two ETA-selective antagonists, BQ-123 and BMS-182874, was reduced 350-2,000-fold. Thus, mutation of a single amino acid in the second transmembrane region of the wild-type ETA receptor results in subtype conversion. In addition, these data represent the first example of peptide interactions with a transmembrane region of a G protein-coupled receptor and indicate that Tyr129, located in the second transmembrane region of the ETA receptor, is a critical component for determination of endothelin receptor subtype-selective ligand binding.

Role of endothelin receptor subtype B (ET-B) in myocardial ischemia.

Previous work indicated that endothelin (ET) may be involved in the pathogenesis of myocardial ischemia, although the relative importance of the ET receptor subtypes is presently not clear. The purpose of this study was to determine the role of myocardial ET-B receptors in mediating ischemic/reperfusion damage in isolated rat hearts. Saturation binding analyses were conducted with [125I]ET-1 and [125I]IRL-1620 to assess changes in ET-A and ET-B receptor binding. Total ET receptor density (Bmax) was greater in atrial versus ventricular tissue. ET-A Bmax was 8 to 10-fold greater than ET-B Bmax. In ischemic and ischemic/reperfused atrial tissue neither the equilibrium dissociation constant (Kd) nor Bmax for ET-B receptors was changed. The ET-B receptor Kd in ischemic or ischemic/reperfused ventricular tissue was also unchanged. In ischemic ventricular tissue there was a trend towards an increased ET-B Bmax, which was accentuated after ischemia/reperfusion. No changes were found in ET-A Bmax or Kd in ischemic ventricular or atrial tissue. The physiological importance of this receptor subtype in ischemic myocardium was determined using the selective ET-B agonist, sarafotoxin S6c. In non-ischemic tissue no effect on coronary flow or function were observed with sarafotoxin S6c. Furthermore, no changes were seen in ischemic time to contracture or any of the reperfusion indexes of myocardial damage. The sarafotoxin S6c utilized was active as it inhibited [125I]ET-3 binding to ET-B receptors (Ki = 0.1 nM). Thus, the pro-ischemic effect of ET-1 seems to be mediated by ET-A receptors. ET-B receptors do not appear to play a role in the pathogenesis of myocardial ischemia.

Expression of endothelin receptor subtypes in rabbit saphenous vein.

Recent investigations have revealed the presence of vasoconstrictory endothelin (ET)-B receptors in several tissues, including the rabbit saphenous vein (RSV). To determine the nature of the ET binding sites in RSV, radioligand-receptor binding studies with selective ligands and Northern analyses with probes from the ET-A and ET-B receptor cDNAs were conducted. ET-1 inhibited 125I-ET-1 binding to RSV in a monophasic manner, with an inhibition constant (Ki) of 0.08 +/- 0.02 nM and a slope factor of 0.9 +/- 0.1. ET-3 inhibition of 125I-ET-1 binding was biphasic, with 68% of the 125I-ET-1 binding sites being displaceable with a Ki value of 31 +/- 4 nM. The remaining 32% of the sites displayed high affinity for ET-3 (Ki = 0.2 +/- 0.1 nM). The ET-A-selective peptide BQ-123 inhibited 125I-ET-1 binding in a biphasic manner, with Ki values of 10.4 +/- 1.9 nM and 3.2 +/- 0.9 microM. The high affinity BQ-123 site comprised 70% of the binding sites, whereas the low affinity site comprised 30%. The correspondence of high affinity binding sites for BQ-123 and low affinity binding sites for ET-3 is consistent with the suggestion that 70% of the 125I-ET-1 binding sites in this tissue are ET-A receptors. To further investigate the nature of the ET-B binding sites in RSV, 125I-ET-3 competition binding experiments were conducted. ET-1 and BQ-123 inhibited 125I-ET-3 binding in RSV with Ki values of 40 +/- 7 pM and 7.2 microM, respectively, whereas inhibition curves for ET-3 and the ET-B receptor-selective agonist sarafotoxin S6c (S6c) were best fit to two-site models. Resultant Ki values for ET-3 and S6c were 50 pM (71%)/4 pM (29%) and 0.7 nM (71%)/95 nM (29%), respectively. Binding in RSV differed from that in rat cerebellum, where ET-3 and S6c inhibition of 125I-ET-3 binding was monophasic (Ki values of 70 pM and 1.1 nM for ET-3 and S6c, respectively). The presence of the nonhydrolyzable guanine nucleotide analog guanosine-5'-O-(3-thio)triphosphate (200 microM) did not affect 125I-ET-3 binding. Low stringency Northern analysis of RSV RNA with [alpha-32P]dCTP-labeled fragments from the ET-A or ET-B receptor cDNAs revealed similar hybridization patterns with both probes, with two resolved RNA species migrating at 4.7 and 1.8 kilobases.(ABSTRACT TRUNCATED AT 400 WORDS)

Control of peptide disulfide regioisomer formation by mixed cysteine-penicillamine bridges. Application to endothelin-1.

While incorporation of penicillamine residues (Pen; beta,beta-dimethyl cysteine) into a peptide can cause dramatic changes in biological activity, the tendency of Pen to form mixed disulfides should also allow the exploitation of the steric bulk of the beta-methyls as a synthetic device to control the production of disulfide isomers. That is, oxidation of a peptide containing an equal number of Cys and Pen residues should predominantly form products which contain mixed Cys-Pen disulfides. Endothelin (ET) is a 21 amino acid peptide which contains Cys at positions 1, 3, 11 and 15. While oxidation of ET tetrathiol has been reported to produce a 3:1 ratio of the natural 1-15, 3-11 to the unnatural 1-11, 3-15 isomers, we show that oxidation of ET analogs containing two cysteines and two penicillamines predominantly formed products containing Cys-Pen disulfides. Random oxidation (air, aqueous NH4OH) of the tetrathiols of [Pen1,11, Nle7]-ET-1 or [Pen3,15, Nle7]-ET-1 produced the correct 1-15, 3-11 isomer in > 12:1 and > 22:1 ratios, respectively. Oxidation of the tetrathiol of [Pen1,15, Nle7]-ET-1 favored the unnatural 1-11, 3-15 isomer by a 4:1 ratio, indicating that a normally contrathermodynamic disulfide isomer can become the favored product as a result of the driving force for penicillamine mixed disulfide formation. Disulfide isomers were identified using ion-spray mass spectrometry in conjunction with enzymatic and acid hydrolysis. [Pen1,11, Nle7]-ET-1 was a partial agonist at the ETA receptor (EC50 = 7.5 nM in rabbit carotid artery rings; Kd = 4.5 nM in rat A10 cell membranes) while [Pen3,15, Nle7]-ET-1 (EC50 = 0.9 nM; Kd = 0.7 nM) was a full agonist with similar potency to ET-1.

Structure-activity studies of endothelin leading to novel peptide ETA antagonists.

With the goal of producing receptor antagonists, numerous monocyclic and bicyclic endothelin analogs were prepared and tested for vasoconstrictor activity, receptor affinity and functional antagonist activity. Bis-penicillamine endothelin analogs containing Ala or Asn at position 18 were functional antagonists, with Ki values of 20-40 nM but KB values of about 1 microM (e.g., [Pen1,11, Nle7, Ala18]-endothelin-1, Ki = 42 nM, KB = 1.2 microM). While these peptides are antagonists at the ETA receptor, they appear to be at least partial agonists at another receptor subtype.

Binding and function of a potent new thromboxane receptor antagonist, BMS 180,291, in human platelets.

Binding and function of BMS 180,291 ([(+)1S-(1 alpha,2 alpha,3 alpha,4 alpha)]-2-[[3-[4-[(n-pentylamino)carbonyl]-2-oxazolyl]-7- oxabicyclo[2.2.1] hept-2-yl]methyl]benzenepropanoic acid]) in human platelets was examined. Kinetic determination of [3H]BMS 180,291 binding produced ligand-receptor association and dissociation rates of 1.4 x 10(7) +/- 0.2 M-1 x min-1 (n = 5) and 0.04 +/- 0.005 min-1 (n = 5), respectively. The resultant Kd was 3.1 +/- 1.1 nM (n = 5). Saturation binding analysis in platelet membranes was consistent with a single class of [3H]BMS 180,291 binding sites with a Kd of 3.6 +/- 0.19 nM (n = 4) and a binding site maxima (Bmax) of 2099.1 +/- 70.3 fmol/mg of protein (n = 4). Specific [3H]BMS 180,291 binding was inhibited by thromboxane A2/endoperoxide receptor antagonists and agonists with a rank order of potency of: BMS 180,291 > or = SQ 29,548 = I-BOP race 15-(1 alpha,2 beta(5Z), 3 alpha(1E,3S),4 alpha) d7-[3-(3-hydroxy-4-(p-iodophenoxy)-1-butenyl)-7- oxabicyclo[2.2.1]hept-2-yl]5-heptenoic acid) > or = BM 13,505 > or = SQ 30,741 = U 44,609 > U 46,619 >> BM 13,177. Prostaglandin E2 and prostacyclin did not appreciably inhibit the specific binding of [3H]BMS 180,291. BMS 180,291 (10 nM-5 microM) shifted the I-BOP-induced platelet shape change curve to the right in a parallel manner without reduction of the maximal response (KB = 13 +/- 3.5 nM; pA2 = 8 +/- 0.2; slope = -1.0 +/- 0.05), whereas 30 nM drug decreased the maximal I-BOP-induced platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of 125I-endothelin-1 and 125I-endothelin-3 in rabbit saphenous vein: evidence for an atypical ET binding component.

Recent investigations have confirmed the presence of vasoconstrictory endothelinB (ETB) receptors in several tissues, including the rabbit saphenous vein (RSV). To determine the molecular nature of the ET receptor subtypes in RSV, radioligand-receptor binding with selective ligands was conducted. ET-1 inhibited 125I-ET-1 binding to RSV in a monophasic manner with an inhibition constant (Ki) of 0.08 +/- 0.03 nM. Inhibition of 125I-ET-1 binding by ET-3 or the ETA-selective peptide BQ-123 resulted in markedly biphasic inhibition curves with Ki values of 0.4 +/- 0.1 nM (36% of total sites)/37 +/- 10 nM (64% of total sites) for ET-3 and 10.4 +/- 1.9 nM (70%)/3.2 +/- 0.9 microM (30%) for BQ-123. The correspondence of high-affinity binding sites for BQ-123 with low-affinity binding sites for ET-3 agrees with the suggestion that 70% of the 125I-ET-1 binding sites in this tissue are ETA receptors. To further investigate the nature of the ET-B (non-ET-A) binding sites in RSV, 125I-ET-3 competition binding was conducted. ET-1 and BQ-123 inhibited 125I-ET-3 binding in RSV with Ki values of 40 +/- 7 pM and 7.2 microM, respectively, while ET-3 and the ETB receptor-selective agonist sarafotoxin S6c (S6c) inhibition curves were best fit to two-site models. Resultant Ki values for ET-3 and S6c were 50 pM (71%)/4 pM (29%) and 0.3 nM (76%)/115 nM (24%).(ABSTRACT TRUNCATED AT 250 WORDS)